Childhood epileptic encephalopathies (EE) are severe neurodevelopmental diseases that manifest in early development. EE is characterized by abnormal electroencephalographic (EEG) activity, intractable seizures comprising of various seizure types, as well as cognitive, behavioral and neurological defects. Developmental and epileptic encephalopathies (DEEs) are a subclass of EEs where the progressive and permanent cognitive and neurophysiological deterioration is not caused by seizure activity alone, but is caused by the same underlying etiology. Recent advances in whole exome sequencing revealed an important role for synaptic dysregulation in DEE and identified multiple new causative variants in synaptic genes. Indeed, mutations in various genes associated with neuronal functions like synaptic transmission and recycling, including transporters, neurotransmitter receptors, and ion channels, have all been identified as causative of DEE. In total, pathogenic DEE-causing variants in over eighty-five genes have been identified and more are likely to follow as next-generation sequencing becomes widely available. DEEs comprise a large group of genetically and phenotypically heterogenous diseases that have been difficult to treat. While in many cases the etiology is unknown, de novo heterozygous missense mutations have often been identified as the underlying cause of DEE. Existing pharmacological interventions by way of antiepileptic drugs leave approximately seventy-percent of DEE patients with intractable seizures. Moreover, these pharmacological treatments do not address the cognitive impairments and associated comorbidities caused by the underlying pathophysiological mechanism. In fact, treatment with antiepileptic drugs may actually worsen cognitive comorbidities due to side effects. Additionally, there are no pharmacological treatments for these cognitive comorbidities other than mood stabilizers and antipsychotics. Therefore, alternative approaches to treatments that address the underlying genetic etiology are necessary. Indeed, the recent utilization of gene therapeutic approaches in other genetic disease models such as spinal muscular atrophy (SMA) has spurred the investigation of gene therapies to treat DEEs. Here, we executed a molecular, behavioral and functional characterization of three preclinical mouse models of DEE involved in synaptic function (Dnm1) and ion channel function (Kcnq3). The human orthologs of the Dnm1 and Kcnq3 genes cause some of the most severe DEE syndromes. Understanding the pathophysiological mechanisms by which mutations in these genes cause disease, is important in identifying and assessing future gene therapeutic interventions. Patients with heterozygous DNM1 pathogenic mutations present with early onset seizures, severe intellectual disability, developmental delay, lack of speech and ambulation, and hypotonia. For the DNM1 dominant-negative model of DEE, we first characterized the Dnm1Ftfl mouse which phenocopies the key disease-defining phenotypes and comorbidities observed in DNM1 patients. Further, we modelled a gene therapy approach in Dnm1Ftfl mice using an RNA interference-based, virally delivered treatment construct. Dnm1Ftfl homozygous mice showed early onset lethality, seizures, growth deficits, hypotonia, and severe ataxia. Molecular analysis of Dnm1Ftfl homozygous mice showed gliosis, cellular degeneration, increased neuronal activation and aberrant metabolic activity, all indicative of recurrent seizure activity. Importantly, our gene therapy treatment significantly rescued all the severe phenotypes associated with DEE, including seizures, early-onset lethality, growth deficits, and aberrant neuronal phenotypes. Thus, our gene therapy approach provided a proof-of-principle for the efficacy of gene silencing to treat DEEs caused by dominant-negative mutations. Second, a DNM1 human variant modelled in mice was generated and characterized. The Dnm1G359A mutation, unlike the Dnm1Ftfl mouse-specific mutation has been identified in patients suffering from DNM1 DEE. Thus, this model allows for a more clinically relevant assessment of the impact of a human DNM1 mutation in mice. In the long run, this model will help validate gene therapeutic approaches that may be clinically relevant to DNM1 DEE patients. The Dnm1G359A mutation, like the Dnm1Ftfl mutation, led to early onset seizures, growth deficits, and lethality, establishing the Dnm1G359A mouse model as a viable model to study DNM1 DEE. In the gain-of-function KCNQ3 model of DEE, Kcnq3R231H mice were characterized molecularly and behaviorally. Patients with KCNQ3 mutations show electrical status epilepticus during sleep (ESES), as well as cognitive and behavioral impairments. The Kcnq3R231H variant led to severe spike-wave discharge phenotype on EEG, decreased maximal seizure threshold, and anxiety-like behavior. Additionally, Kcnq3R231H led to increased localization of Kcnq3 protein at neuronal membranes, suggesting a role for membrane aggregation on disease phenotypes. Altogether, these findings show the viability of preclinical models of both dominant-negative and gain-of-function mutations in replicating key disease-defining phenotypes associated with severe DEEs. Additionally, the results presented here establish a proof-of-principle demonstration that gene silencing can rescue severe phenotypes caused by dominant-negative mutations in DEE. Future studies on both dominant-negative and gain-of-function models should enable an in-depth understanding of mechanistic implications for each mutation, and lead to gene therapeutic strategies to mitigate the debilitating phenotypes of these DEEs.

Cytoplasmic dynein has been implicated in a wide range of functions. Originally characterized as being responsible for retrograde axonal transport, its has also been shown to traffic vesicular organelles (Golgi, endosome and lysosome distribution), transport viral particles to the nucleus, and participate in microtubule organization. During mitosis, cytoplasmic dynein is thought to function in spindle pole focusing and prometaphase kinetochore capture. This thesis explores the mitotic roles of cytoplasmic dynein. The first chapter addresses the role of cytoplasmic dynein in kinetochore activity. Using immunofluoresence microscopy, a number of motors and related proteins were observed at the primary, but not secondary, constrictions of prometaphase multicentric chromosomes. The proteins assessed included the cytoplasmic dynein intermediate chains, three components of the dynactin complex (dynamitin, Arp1, and p150Glued), the kinesin related proteins CENP-E and MCAK, and the proposed structural and checkpoint proteins CENP-F, HZW10, and MAD2. The differential localization of these proteins offered new insight into the assembly and composition of both active and inactive centromeres, and provided a molecular basis for the apparent inactivity of the latter during chromosome segregation. The second chapter characterizes LIS1, a protein that is defective in the developmental brain disease type1 lissencephaly. Mutations in the LIS1 gene cause gross histological disorganization of the developing cerebral cortex resulting in a nearly smooth brain surface. Because genetic evidence from lower eukaryotes suggested that LIS1 acted within the cytoplasmic dynein pathway, it was of interest to analyze LIS1 in terms of cytoplasmic dynein function. LIS1 was found to coimmunoprecipitate with cytoplasmic dynein and its companion complex dynactin. During mitosis LIS1 localized to the prometaphase kinetochore, spindle microtubules and the cell cortex, known sites for cytoplasmic dynein binding. Interference with endogenous LIS1 in cultured mammalian cells displaced dynein localization and disrupted mitotic progression. LIS1 was concluded to participate in cytoplasmic dynein functions, but only during mitosis. Data presented in the final chapter further characterizes LIS1's interactions with microtubules, cytoplasmic dynein and the mammalian homologue of NUDC. LIS1 was not found to co-fractionate with microtubules, nor did overexpression of LIS1 cause visible effects on microtubule organization or dynamics. GFP-LIS1 was shown to ride along the plus ends of growing microtubules. Though LIS1 was not found to have a direct effect on microtubules, it may regulate dynein's microtubule binding activity. LIS1 was found to co-immunoprecipitate with a co-overexpressed cytoplasmic dynein subunit substantiating the existence of a dynein LIS1 supercomplex. Furthermore, association of these proteins increased markedly in mitotically blocked samples. LIS1's regulation of cytoplasmic dynein may change the capacity of the motor to efficiently manipulate its mitotic cargoes, dramatically effecting the timing of cell division. This disruption has implications for the fundamental role of cytoplasmic dynein during early embryonic development.

No description available.

Double-stranded RNA (dsRNA) is a key molecule that initiates the immune response to viral infection, but increasingly endogenous (self) dsRNA has been found to be central to the pathology of diverse non-infectious diseases, from neurodegenerative disease to autoimmunity to cancers. Therefore, it is critical to understand the mechanisms that regulate endogenous dsRNA and the pattern recognition receptors that sense dsRNA. In this dissertation, I address three main questions pertaining to this. First, why is the brain so prone to dsRNA-mediated non-infectious disease, especially considering that dsRNA sensors are expressed in almost all tissues. Using stem cell differentiation, gene expression manipulation, and microscopy, I determined that neurons are a special cell type that express high levels of endogenous dsRNA. This high dsRNA burden in neurons is driven by global lengthening of 3`untranslated regions (3`UTRs) and induces tonic inflammation. Second, I examined the mechanism through which the dsRNA regulator ADAR1 controls endogenous dsRNA levels. I employed heavy use of tissue culture and visualization of dsRNA by confocal microscopy to determine that both the dsRNA-binding and dsRNA-editing activities of ADAR1 are required to suppress global endogenous dsRNA levels. Third, after identifying the existence of transcript isoforms of the key dsRNA sensor PKR, I explored their regulatory potential on the PKR protein itself. By genetically altering human cells, I identify that 3`UTR isoforms of PKR regulate transcript localization, translation efficiency, and PKR protein activatability. Overall, the studies described herein demonstrate novel regulatory roles of endogenous dsRNA and underscore the importance of dsRNA in neurological disease.

雖然由老化引發的腦白質病變是老年人認知障礙的一個重要誘因，其機理缺並不為人所知。最新的小樣本研究表明擴散核磁造影在很大程度上是對腦白質病變最為敏感的的成像檢測手段。加深對擴散核磁造影所給出的各種指數的理解和認知對於檢測腦白質病變的病理發展以及研發試驗療法的替代標記有重要的意義。 / 為了獲得更具有臨床價值的擴散核磁造影指數，我們首先需要重構腦白質纖維束並沿著重構出的腦白質纖維束採集數值。然而，傳統的腦白質纖維束重構技術對於腦白質病變十分敏感。此外，不同病人所重構出的腦白質纖維束間缺乏映射關係也使我們無法有效進行大樣本統計分析。 / 在這個課題裡，我們提出了一個可以解決以上問題的一個全新框架。我們將專家標註過功能區的全腦白質纖維束模板配準到各個個體的腦部。此方案可自動生成個體化的全腦白質纖維束以及纖維束的功能區標註。自由形變模型被用於在全局層面對配準進行約束。所重構纖維束的曲率被用於在局部對配準進行約束。為了減輕腦白質病變對配準的影響，我們運用了一種 魯棒的主成分分析手段來檢測被病灶所干擾的纖維束。為了指導這些被干擾纖維束的配準，我們提出了一種全新的沿纖維束的區域特徵作為替代。此外，我們也探究了通過在纖維束上建立坐標系來除去離群纖維已經提供更高相關性的辦法。 / 我們所提出的框架被運用於一個腦小血管病變的臨床研究。在64個研究對象中約半數是腦白質病變患者。試驗結果證實此算法成功地將全腦白質纖維束模板配準到了所有研究對象上。沿著特定纖維束改採集的指數與認知測試分數的相關性顯著地超越了傳統指數所給出的結果。我們同時也發現沿著不同功能區腦白質纖維改採集的指數與相應的認知測試分數統計相關。 / Although age-related white matter lesion(WML)is an important substrate for cognitive impairment in the elderly, the mechanisms whereby WML induces cognitive impairment are uncertain. Recent findings based on small studies suggested that diffusion tensor imaging (DTI) measures might be the most sensitive imaging predictors in patients with WML. Understanding the imaging predictors for such disease will be useful in monitoring disease progression and in devising surrogate marker for treatment trials. / In order to obtain DTI measurements with diagnostic significance, it is first necessary to reconstruct the white-matter fiber pathways inside the brain along which certain DTI-derived values are calculated. Nevertheless, the traditional approach of white-matter tract reconstruction, or tractography, is severely hindered by the abundant existence of lesions inside the brains of WML patients. The lack of correspondence between fiber bundles across patients also makes obtaining group statistics of individual fiber bundles dicult. / In this study, we propose a novel framework that can mitigate the aforementioned issues of traditional tractography approaches. An expert-labeled whole brain tractography template is registered onto individual patients. Fiber trajectories and anatomically meaningful fiber bundles are automatically obtained by this registration. The free-form deformations are used to regularize the transformations at the whole brain level and across fiber bundles. Fiber curvatures are penalized as the intra-fiber regularization to encourage the smoothness of transformed fibers. White matter (WM) lesion is one of the major factors affecting tractography and registration of subjects with neuro Logical disorders. The Robust Principal Component Analysis(RPCA) is used to automatically detect fiber tract segments that are affected by WM lesion and a novel along-fiber regional prior is learned from healthy subjects to facilitate the registration of these fiber tract segments. We also propose to establish bundle-wise coordinate system by utilizing low-rank constraints upon the DTI measurements. The eort elevates the summary for an anatomical bundle from a scalar statistic to a vector containing changes along the representative fiber pathway. It provides means to exclude the outlier fibers while retaining partially-complete fibers. / The proposed scheme is applied to a clinical study of cerebral small vessel diseases(SVD).Experimental results show successful registration of the whole brain tractography template onto all 64 subjects, including both healthy con¬trol subjects and SVD patients. The DTI measures measured along specific registered anatomical fiber bundles exhibit significant boost in correlation with cognitive functions as compared with traditional measures. It also shows that different anatomical WM structures correlate with multiple types of cognitive functions in different ways. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / He, Xiaotian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 46-53). / Abstracts also in Chinese. / List of Figures --- p.ix / List of Tables --- p.xii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Motivation --- p.1 / Chapter 1.2 --- Our Work and Contributions --- p.2 / Chapter 1.3 --- Related Work --- p.4 / Chapter 1.4 --- Thesis Organization --- p.5 / Chapter 2 --- Background --- p.6 / Chapter 2.1 --- Background of Neuroanatomy --- p.6 / Chapter 2.2 --- Background on Diffusion Tensor Magnetic Resonance Imaging (DTMRI) --- p.11 / Chapter 3 --- Free Form Fibers --- p.18 / Chapter 3.1 --- DTI Acquisition --- p.20 / Chapter 3.2 --- Fiber Model --- p.20 / Chapter 3.3 --- Fiber-to-DTI Registration --- p.21 / Chapter 3.3.1 --- Free-Form Fibers (FFFs) --- p.21 / Chapter 3.3.2 --- Tensor-Driven Fiber-to-DTI Registration --- p.23 / Chapter 3.3.3 --- Reliability Assessment by Robust Principal Component Analysis --- p.24 / Chapter 3.3.4 --- Contextual Feature --- p.26 / Chapter 3.3.5 --- Learning the Fiber Context Prior --- p.29 / Chapter 3.3.6 --- Registration Refinement Using the Fiber Context Prior --- p.29 / Chapter 4 --- Results --- p.31 / Chapter 5 --- Future Work --- p.39 / Chapter 5.1 --- Refinement on Large Bundles --- p.39 / Chapter 5.2 --- Outlier Fiber Removal in Fiber Template --- p.40 / Chapter 6 --- Conclusion --- p.44 / Bibliography --- p.46

Thesis (M. Tech. (Clinical technology )) - Central University of technology, Free State, 2013 / The incidence of epilepsy is high in sub-Saharan Africa and resource poor countries (RPCs). There are few neurologists and paediatric neurologists to manage people with epilepsy (PWE). Health care is often limited, particularly technological services, including electroencephalogram (EEG), video EEG monitoring, and Neuroradiology services. All these are important in the management of PWE. Since 2008, informal electrophysiology training has been provided at the Red Cross War Memorial Hospital, in the Department of Paediatric Neurology. The Principal Investigator (PI) elected to develop a formal teaching course on EEG interpretation at the Red Cross War Memorial Hospital. A study was designed to evaluate the practical use of a handbook entitled “Handbook of Paediatric Electroencephalography: A guide to basic paediatric electroencephalogram interpretation.” This has been developed to fulfill the need for basic understanding and interpretation of EEG amongst clinicians caring for children in sub-Saharan Africa who may not have access to, or be able to afford, training at a recognized facility or on-line. In 2008, the department of Paediatric Neurology at the Red Cross War Memorial Hospital had their first African fellow from Kenya. By 2011, seven participants had undergone EEG training. A quantitative research approach and design was used in order to evaluate the handbook in terms of the accessibility of the contents and its practical use. Quantification included the recruitment of participants who constituted the population sample, a pilot study, and the collection of data from comparative assessments of participants’ use of the handbook, and from questionnaires completed by participants. This provided the researcher with the opportunity to improve and validate her knowledge of training in EEG interpretation. The researcher was able to quantify and compare the scores of participants using the handbook, as well as to compare their evaluative responses to its content and practical use. Eleven of thirteen participants completed the study. The pre-training results showed a median percentage of 50 which increased to 70 percent post-test. A comparison of the scores of trained versus not-trained revealed that those participants who had undergone one-on-one training on site at the unit fared much better both in their interpretations, conclusions, and reporting of EEG findings. The responses from the evaluative and comparative survey between the two groups showed no significant difference across all questions, the majority of the questions on the relative usefulness of the handbook being rated ‘agree’ and ‘strongly agree’, thus supporting the finding that all participants found the handbook useful whether they had received one-on-one training or not. The post-training results in EEG interpretation showed a stronger trend towards statistical significance (p<0.06) with trained participants and with the not-trained. These findings lend support to the success and usefulness of the handbook as a basic guide to paediatric EEG interpretation. The handbook was not aimed at making the electroencephalography reader an expert at a specialist level, but rather to maximize the reliability of the reading of EEG when screening electroencephalograms for important key diagnostic markers which would alter the child’s management. This is the first published handbook on paediatric EEG in South Africa. The results of this study strongly suggest that the handbook is useful as a learning and reference tool in interpretation of paediatric EEG, both for individuals with access to one-on-one training as well as those without. It is intended that the handbook, in conjunction with one-on-one training, will form part of a post-graduate diploma course offered by the University of Cape Town on “basic electrophysiology and the management of children with epilepsy” for training neurologists and child neurologists, paediatricians and health care workers in sub-Saharan Africa.

University of Macau / Institute of Chinese Medical Sciences

As demências Frontotemporais (DFT) compreendem 2 fenótipos clínicos: distúrbios comportamentais ou de linguagem. Coletivamente, as DFT podem ser causadas por um grupo diversas de doenças neurodegenerativas chamadas degeneração lobar frontotemporal (DLFT). Novas entidades têm sido descritas neste grupo e o conceito está em constante evolução. Parte dos mecanismos envolvidos na morte celular nas DLFTs também são observados n envelhecimento normal. Determinar as entidades e freqüência das DLFTs em uma série com utilização de imunoistoquímica. Uma série prospectiva de 833 casos avaliados prospectivamente no Centro de Pesquisas de Doença de Alzheimer da Washington University - EUA. Os casos de DFT foram selecionados por critérios clínicos e a classificação neuropatológica foi baseada em protocolos universalmente aceitos para DLFT. Dos casos de demência, 53(6,3%) atenderam aos critérios clínicos e neuropatológicos para DLFT. Outros 8 casos atenderam apenas aos critérios clínicos de DFT. As tauopatias representaram 40% dos casos. Entretanto, a maioria dos casos apresentava inclusões ubiquitina-positivas e tau-negativas. Esclerose hipocampal e alterações do tipo Doença de Alzheimer foram encontradas em 12 e 10 casos, respectivamente. Apesar da DLFT-U ter sido a entidade mais freqüente nesta série, entidades e menos comuns não incluídas nas recomendações de McKhann também podem apresentar fenótipo clínico de DFT. A inclusão destas novas entidades é mais uma evidência de que os sintomas clínicos são mais dependentes das áreas acometidas do que da entidade em si. A melhor compreensão desses mecanismos tem um grande potencial em auxiliar no desenvolvimento de medidas que possam modular ou retardar os efeitos do envelhecimento no cérebro, além é claro de trazer possibilidade de tratamento, hoje inexistente, para os pacientes acometidos. / Frontotemporal dementia (FTD) encompasses two clinical phenotypes: progressive behavioral change and language disorder. Collectively, FTD may be caused by a diverse group of neurodegenerative diseases called frontotemporal lobar degenerations (FTLDs). New entities have been described and the nosology of FTLDs continues to evolve. To determine the type and frequency of FTLDs in a series using contemporary immunohistochemical methods. Eight hundred and thirtythree dementia cases were prospectively assessed at Washington University Alzheimer Disease Research Center (WUADRC) and cases with clinical FTD were identified using existing diagnostic criteria and neuropathologic entities were ascertained using immunohistochemistry and contemporary diagnostic criteria. Of the dementia cases, 53(6.3%) met clinical criteria for FTD; 45(5.1%) fulfilled both clinical and neuropathological criteria for FTLD, and another 8 fulfilled only the clinical criteria. Forty percent of the cases were tauopathies. However, most FTLD cases were characterized by ubiquitin-positive, tau-negative inclusions. Co-existing hippocampal sclerosis and AD-type changes were observed in 12 and 10 cases, respectively. Although FTLD-MND-type is the most frequent FTLD in this prospectively assessed series, less common entities not included in the McKhann criteria, may also present clinically as FTD and should be considered as part of the neuropathologic spectrum of FTLDs that may be encountered in the dementia clinic. The better understanding of the cell death mechanisms related to those entities is likely to contribute for the development of a treatment for FTLD as well for a way of modulate brain aging.

Brain diseases including neurological disorders and tumors remain undertreated due to the challenge in accessing the brain, and blood-brain barrier (BBB) restricting drug delivery, which also profoundly limits the development of pharmacological treatment. Focused ultrasound (FUS) with acoustic agents including microbubbles and nanodroplets remains as the only method to open the BBB noninvasively, locally, and transiently to assist drug delivery. For an ideal medical system to serve a broad patient population, it requires precise and flexible targeting with simulation to personalize treatment, real-time monitoring to ensure safety and effectiveness, and rapid application, as repetitive pharmacological treatment is often required. Since none of current systems fulfills all the requirements, here we designed a neuronavigation-guided FUS system with protocol assessed in in vivo mice, in vivo non-human primates, and human skulls from in silico preplanning, online FUS treatment and real-time acoustic monitoring and mapping, to post-treatment assessment using MRI. Both sedate and awake non-human primates were evaluated with total treatment time averaging 30 min and 3-mm targeting accuracy in cerebral cortex and subcortical structures. The FUS system developed would enable transcranial FUS in patients with high accuracy and independent of MRI guidance.

Thesis (doctoral)--Universitetet i Lund. / Reprints of journal articles inserted in pocket inside back cover.